of April 3, 2019.This information is current as

Interaction with CCR2Orthologs Induce Chemotaxis through

-Defensin 2 and 3 and Their MouseβHuman

HehlgansJohann Röhrl, De Yang, Joost J. Oppenheim and Thomas

http://www.jimmunol.org/content/184/12/6688doi: 10.4049/jimmunol.0903984May 2010;

2010; 184:6688-6694; Prepublished online 17J Immunol 

Referenceshttp://www.jimmunol.org/content/184/12/6688.full#ref-list-1

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The Journal of Immunology

Human b-Defensin 2 and 3 and Their Mouse OrthologsInduce Chemotaxis through Interaction with CCR2

Johann Rohrl,* De Yang,* Joost J. Oppenheim,* and Thomas Hehlgans†

b-defensins play a dual role during immune response. Their direct antimicrobial properties contribute to the local innate immune

response by combating microbial invasions. Furthermore, previous studies revealed the capacity of certain b-defensin family

members to chemoattract immature dendritic cells and CD45RO+ CD4+ T cells through chemokine receptor CCR6. However,

because b-defensins also chemoattract macrophages and monocytes, which do not express CCR6, efforts have been made to

identify other receptors for these polypeptides. In this study, we demonstrate the capacity of human b-defensin (hBD)2 and 3 and

their mouse orthologs, b-defensin 4 and 14, to interact with CCR2, a chemokine receptor expressed on monocytes, macrophages,

and neutrophils. These b-defensins, fused to the Fc region of human IgG1, showed binding to CCR2-transfected HEK293 cells, as

revealed by flow cytometry. The b-defensin fusion proteins also induced CCR2-specific chemotaxis of transfected HEK293 cells,

human peripheral blood monocytes, and mouse peritoneal exudate cells in a dose-dependent manner. Preincubation of human

monocytes with CCL2/MCP-1, the chemokine ligand for CCR2, abolished migration induced by b-defensins. Conversely, pre-

incubation with hBD2:Ig or hBD3:Ig inhibited MCP-1 induced migration. Peritoneal exudate cells from CCR2-deficient mice

failed to migrate toward these fusion proteins. In conclusion, the b-defensins used in this study contribute to the innate and

adaptive immune response in their role as chemoattractants. Our data indicate that hBD2 and hBD3, together with their mouse

orthologs (b-defensin 4 and 14), are chemotactic for a broad spectrum of leukocytes in a CCR6- and CCR2-dependent manner.

The Journal of Immunology, 2010, 184: 6688–6694.

The family of b-defensins consists of a variety of cationicantimicrobial polypeptides that contribute to the immuneresponse against bacterial, fungal, and viral infections (1).

Their tertiary structure is composed of a N-terminal a helix, fol-lowed by a b-sheet, and contains three family-specific disulfideconnectivities between cysteines 1–5, 2–4, and 3–6 (2–4). Theexpression of b-defensins is induced by different pathogen-associated molecular patterns, such as LPS, bacterial lipoprotein,and other TLR and NODR ligands (5–7). Furthermore, distinctcytokines, such as IFN-g, TNF, and IL-1b, IL-17, and IL-22, canalso trigger b-defensin expression (8–11).In addition to their antimicrobial effects within the innate im-

mune system, b-defensins contribute to the adaptive immune re-sponse. It was shown that mouse b-defensin (mBD)2 activatesimmature dendritic cells through TLR4, triggering a Th1 milieu(12). Human b-defensin (hBD)3 activates APCs via TLR1 and

TLR2 in a NF-kB–dependent manner (13). In earlier reports, weand other investigators described the capacity of hBDs (e.g., 1–3)(14, 15) or mBDs (e.g., 2 and 14) (16, 17) to induce chemotaxisthrough CCR6. Recently, it became evident that Cys5 playsa crucial role in CCR6-dependent chemotaxis (18). However,there is also clear evidence for the chemotactic interaction ofb-defensins with another pertussis toxin sensitive Gi protein-coupled receptor expressed on monocytes and macrophages ormast cells, which do not express CCR6 (15, 19–21).Tumor-derived hBD3 uses CCR2 to attract tumor-infiltrating

monocytes (G. Jin, personal communication). It was reported thathBD3 inhibits CXCR4-dependent chemotaxis and calcium mobi-lization induced by CXCL12/stromal cell-derived factor-1a (SDF-1a), and it induces the internalization of CXCR4, indicating aninteraction of hBD3 with this chemokine receptor (22).In this study, however, we demonstrated the capability of b-

defensins to bind to the chemokine receptor CCR2, rather thanCXCR4, and to subsequently induce chemotaxis. CCR2 is expressedon monocytes, dendritic cells, and macrophage subsets (23, 24),and two of its well-characterized ligands are CCL2/MCP-1 andCCL7/MCP-3 (25–27). The interaction of MCP-1 and -3 withCCR2 plays a pivotal role during the immune reaction against bac-terial or viral infections, attracting CCR2-expressing inflammatorycells to the sites of infection (23, 26, 28).In this article, we provide data that substantiate the role of b-

defensins as chemoattractants recruiting CCR6+ and CCR2+ cells.

Materials and MethodsAnimals

Wild-type C57BL/6 mice were purchased from Charles River Laboratories(Frederick, MD). CCR2-deficient mice (29) on a C57BL/6 backgroundwere bred in the Animal Production Area of the National Cancer Institute.All mice were kept under specific pathogen-free conditions, with water andfood given ad libitum. All experiments with mice were performed incompliance with the principles and procedures outlined in the National

*Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program,Division of Basic Science, Center for Cancer Research, National Cancer Institute,Frederick, MD 21702 and †Institute of Immunology, University of Regensburg,Regensburg, Germany

Received for publication December 11, 2009. Accepted for publication April 12,2010.

This work was supported in whole or in part with federal funds from the NationalCancer Institute, National Institutes of Health, under contract HHSN261200800001E;in part by the Intramural Research Program of the Center for Cancer Research, NationalCancer Institute, National Institutes of Health; and by Grant PDOK-62-08 from theBayerische Forschungsstiftung.

The content of this publication does not necessarily reflect the views or policies of theDepartment of Health and Human Services, nor does mention of trade names, com-mercial products, or organizations imply endorsement by the U.S. Government.

Address correspondence and reprint request to Prof. Dr. Thomas Hehlgans, Instituteof Immunology, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93042Regensburg, Germany. E-mail address: thomas.hehlgans@klinik.uni-regensburg.de

Abbreviations used in this paper: hBD, human b-defensin; mBD, mouse b-defensin;PEC, peritoneal exudate cell; SDF-1a, stromal cell-derived factor-1a; shBD, synthetichuman b-defensin.

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Institutes of Health Guide for the Care and Use of Animals and wereapproved by the National Cancer Institute Frederick Animal Care andUse Committee.

Expression and purification of b-defensin fusion proteins

rb-defensin fusion proteins were generated and purified as described ear-lier (17). In brief, cDNA encoding for the mature polypeptides of hBD2,hBD3, mBD4, or mBD14 was cloned into the Signal Ig plus vector (R&DSystems, Wiesbaden, Germany). All cDNAs fused to the human IgG-Fcsequence were subcloned into the pMTBiP/V5-His A expression vector(Invitrogen, Karlsruhe, Germany), containing a hygromycin cassette, byPCR amplification of the hBD2:Ig, hBD3:Ig, mBD4:Ig, and mBD14:IgcDNAs, using the following primers: 59-CCC AGA TCT GTT ACGTGC CTG AAA AGC GG-39 for hBD2-59; 59-CCC AGA TCT CAGAAA TAC TAC TGC CGT G-39 for hBD3-59; 59-CCC AGA TCT AATCCA ATA ACA TGC ATG-39 for mBD4-59; 59-CCC AAG CTT CGAAAA TTT TTC TGC AGA-39 for mBD14-59; and 59-CG CGG CCGCCA TCA TTT ACC CGG AGA CAG G-39 for human IgG-Fc-39. Aftertransfection, stable expressing Drosophila-S2 cells were selected andmaintained in hygromycin (0.3 mg/ml; Invitrogen). The b-defensin fusionproteins were purified from the culture medium using HiTrap Protein G HPcolumns (GE Healthcare, Munich, Germany), according to the manufac-turer’s instructions. Expression and purification of the fusion proteins wereconfirmed by Western blotting using peroxidase-conjugated donkey anti-human IgG mAb (Dianova, Hamburg, Germany) and polyclonal Absagainst hBD2, hBD3, and mBD4 (Santa Cruz Biotechnology, Heidelberg,Germany).

Cell isolation and culture

HEK293 cells expressing human CCR2 variant B (hCCR2/HEK293) andHEK293 cells expressing human CXCR4 (hCXCR4/HEK293) weremaintained in DMEM (Mediatech, Herndon, VA) containing 10% FCS(HyClone, Thermo Scientific, Logan, UT; inactivated for 30 min at 56˚C)and 800 mg/ml G418 and were harvested when they reached 70–80%confluence.

Human peripheral blood enriched in mononuclear cells was obtainedfrom healthy donors by leukapheresis (Transfusion Medicine Department,Clinical Center, National Institutes of Health, with approved human subjectagreement). The blood was centrifuged through Histopaque-1077 (Sigma-Aldrich, St. Louis, MO), and PBMCs collected at the interface were washedwith PBS. After centrifugation through an iso-osmotic Percoll (GE Health-care, Pittsburgh, PA) gradient, the enriched monocytes (peripheral bloodmonocytes) were obtained from the top of the gradient.

Mouse peritoneal exudate cells (PECs) were elicited by i.p. injection of2 ml 3% thioglycollate into 8–12-wk-old C57BL/6 wild-type or CCR2-deficient mice. After 3 d, PECs were isolated by lavage of the peritoneumwith 5 ml ice-cold PBS containing heparin (20 U/ml) and EDTA (5 mM).

Chemotaxis assay

Cells were suspended in chemotaxis medium (RPMI 1640 containing 1%BSA, 20 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, and 100mg/ml streptomycin) at 1 3 106 cells/ml for human monocytes, 5 3 105

cells/ml for PECs, and 2 3 106 cells/ml for human CCR2+, humanCXCR4+, or wild-type HEK293 cells. In certain experiments, primarycells were preincubated with 10–100 ng/ml chemoattractant (as indicated)for 30 min at 37˚C in humidified air containing 5% CO2. Synthetic hBD2and hBD3 were purchased from Peptide Institute (Osaka, Japan). Humanor mouse CCL2/MCP-1 and human CXCL12/SDF-1a were purchasedfrom PeproTech (Rocky Hill, NJ) and used as positive control. Themigration of cells in response to chemoattractants (b-defensins or controlchemotactic factors) was determined using the 48-well microchemotaxischamber assay, as previously described (30). In brief, chemoattractantsdiluted in chemotaxis medium at various concentrations were put into thelower wells of a 48-well microchemotaxis chamber (NeuroProbe, Gai-thersburg, MD), and cell suspension was added to the upper wells. Thelower and upper compartments were separated by an uncoated polycar-bonate filter membrane (5 mm pore size) for primary cells or a collagen-coated polycarbonate filter membrane (10 mm pore size) for humanCCR2+, human CXCR4+, or wild-type HEK293 cells (both from NeuroP-robe). After incubation at 37˚C for 1.5 h for primary cells and 5 hfor HEK293 cells in humidified air with 5% CO2, the filters were re-moved, scraped, and stained. The cells that migrated across the filterwere counted under a light microscope using Bioquant Life Science soft-ware (Bioquant Image Analysis, Nashville, TN). The results (mean 6SD of triplicate wells) are presented as the number of cells per high-power field.

Flow cytometry

All staining steps were performed for 20 min on ice in PBS containing 2%FCS and 0.05% Azide. HEK293 cells were incubated with 10% goat serumto prevent nonspecific binding. Primary cells were blocked with 10% goat orrat serum. HEK293 cells were labeled with 1 mg/ml b-defensin fusionprotein. After washing, cells were stained with a PE-conjugated anti-human IgG Ab (Jackson ImmunoResearch Laboratories, West Grove,PA). Expression of CCR2 or CCR6 on human monocytes was analyzedusing an anti-human CCR2-PE Ab (clone 48607) or an anti-human CCR6-FITC Ab (clone 53103) purchased from R&D Systems (Minneapolis,MN). Detection of CCR2 or CCR6 expression on PECs was performedusing an anti-mouse CCR2 Ab from goat (GeneTex, Irvine, CA) and ananti-goat IgG-FITC secondary Ab (Jackson ImmunoResearch Laborato-ries) or an anti-mouse CCR6 Ab from rat (clone 140706; R&D Systems)and an anti-rat IgG-Alexa 488 Ab (Molecular Probes, Eugene, OR). Flowcytometry was performed on a FACScan cytometer using Cell Quest soft-ware (BD Biosciences, San Jose, CA). Flow cytometry data were analyzedwith FlowJo software (version 8.1.0) (Tree Star, Ashland, OR).

Statistical analysis

Statistical analyses of chemotaxis data were performed using one-wayANOVA with the Dunnett posttest (GraphPad Prism, version 4.0c; Graph-Pad, San Diego, CA); medium control served as the reference value. Sta-tistical significance was considered at p , 0.01.

ResultsBinding of b-defensins to CCR2 but not CXCR4

The b-defensins used in this study were expressed as fusion pro-teins, fused to the Fc region of human IgG1 (Fig. 1A), allowingeasy detection of binding. Expression of fusion proteins wasdetected by Western blot analysis using an anti-human IgG Ab(Fig. 1B, lower panels). The b-defensin domain of the fusionproteins was detected by polyclonal Abs against hBD2, hBD3,and mBD4 (Fig 1B, upper panels). Both detection methodsrevealed an apparent m.w. of 37 kDa for the b-defensin fusionproteins (Fig. 1). There is no commercially available Ab againstmBD14. In earlier studies, we showed that these b-defensin fusionproteins retained antimicrobial activity comparable to unfusedb-defensins (17, 31).In this study, hBD2:Ig and hBD3:Ig, as well as their mouse

orthologs mBD4:Ig and mBD14:Ig, were used to examine theirinteraction with CCR2 and CXCR4. FACS analysis revealedthat hBD2:Ig, hBD3:Ig, mBD4:Ig, and mBD14:Ig bound to hu-man CCR2-expressing HEK293 cells independently of species

-defensin hIgG1 (Fc)

hBD2:Ig

hBD3:Ig

mBD4:

Ig

mBD14

:Ig

37 kDa

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37 kDa anti-

-defensin

anti-hIgG1

FIGURE 1. Expression of hBD2, hBD3, and their mouse orthologs

(mBD4 and mBD14) as human IgG1-Fc fusion proteins. A, Schematic

representation of the b-defensin fusion protein (not to scale). B, Western

blot analysis of recombinantly expressed hBD2:Ig, hBD3:Ig, mBD4:Ig,

and mBD14:Ig using polyclonal Abs against hBD2, hBD3, and mBD4

(upper panels) or anti-human IgG1 Ab (lower panels). Data are represen-

tative of at least three independent experiments.

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specificity (Fig. 2A). In contrast, no binding of b-defensin fusionproteins was observed using human CXCR4-expressing HEK293cells (Fig. 2B). Unfused human IgG1 did not bind to wild-type ortransfected HEK293 cells (Fig. 2).

The b-defensins induced chemotaxis of humanCCR2-expressing HEK293 cells

Chemotaxis assays using human CCR2-expressing HEK293 cellswere performed to elucidate whether the binding of hBD2:Ig andhBD3:Ig, as well as mBD4:Ig and mBD14:Ig, to human CCR2 hasfunctional consequences. Human MCP-1 induced dose-dependentmigration of human CCR2-expressing HEK293 cells in the typicalbimodal manner, reaching a maximum at a concentration of 10ng/ml (Fig. 3A). Human SDF-1a, the ligand for human CXCR4,induced dose-dependent migration of human CXCR4-expressingHEK293 cells, with maximal migration at 100 ng/ml (Fig. 3B).The b-defensin fusion proteins induced chemotaxis of humanCCR2-expressing HEK293 cells in a dose-dependent manner,which peaked at a concentration of 100 ng/ml (Fig. 3C–F). In agree-ment with the inability of the b-defensin fusion proteins to bind tohuman CXCR4+ HEK293 cells, they also failed to induce chemo-taxis of these cells (Fig. 3C–F). To exclude effects related to thehuman IgG1-Fc part of the fusion proteins, we tested unfused humanIgG1 and showed that it did not induce cell migration (Fig. 3G).

The b-defensin fusion proteins induced chemotaxis of humanperipheral blood monocytes

Human monocytes isolated from peripheral blood, which expressendogenous CCR2 but not CCR6 (Fig. 4), were used to determinewhether the b-defensin fusion proteins were also chemotactic forprimary monocytes. Similar to the migration induced by humanMCP-1 (Fig. 5A), the chemotactic effect of hBD2:Ig and hBD3:Ig,as well as mBD4:Ig and mBD14:Ig, peaked at a concentration of10 ng/ml (Fig. 5C–F). In contrast, monocytes preincubated with10 ng/ml human MCP-1 failed to migrate toward the b-defensinfusion proteins (Fig. 5C–F). Conversely, preincubation of mono-cytes with 10 ng/ml hBD2:Ig or hBD3:Ig similarly induced de-sensitization and inhibited subsequent chemotactic responsestoward human MCP-1 (Fig. 5A). However, these preincubated

cells retained their ability to migrate toward human SDF1-a(data not shown). Human monocytes failed to migrate towardhuman IgG1 (Figs. 5B, 6). The chemotactic effect of hBD2:Igand hBD3:Ig, at concentrations of 10 ng/ml, was comparable tothat of synthetic hBD (shBD)2 and shBD3 for human monocytes(Fig. 6). Pretreatment of monocytes with 10 ng/ml shBD2inhibited hBD2:Ig-induced migration (Fig. 6A). Similarly, pre-treatment with 10 ng/ml shBD3 abolished hBD3:Ig-induced che-motaxis (Fig 6B). Synthetic mBD4 and mBD14 were notcommercially available for further analysis.

CCR2-dependent migration of PECs

Thioglycollate-elicited PECs from C57BL/6 wild-type mice area heterogeneous cell population. The majority of PECs expressedCCR2 (Fig. 7A). CCR6-expressing cells were not detected (Fig.7B). Mouse MCP-1 induced maximal migration at a concentrationof 1 ng/ml (Fig. 8A). However, the PECs also showed chemotacticresponses to mBD4:Ig, mBD14:Ig, and hBD2:Ig, peaking at a con-centration of 100 ng/ml (Fig. 8C–E). Furthermore, hBD3:Ig in-duced maximum migration at a concentration of 10 ng/ml (Fig.8F). The capacity to migrate toward b-defensin fusion proteins ormouse MCP-1 was abolished in PECs from CCR2-deficientC57BL/6 mice (Fig. 8A, 8C–F). Unfused human IgG1 did notinduce migration of PECs (Fig. 8B).

DiscussionIn earlier reports, we described the production and characterizationof b-defensin fusion proteins. These fusion proteins retained theirantibacterial and chemotactic activities (17, 31) and were chemo-tactic for CCR6-expressing HEK293 cells, as well as for mono-cytes that do not express CCR6. The constant human IgG1 domainof the fusion proteins facilitated efficient purification and detec-tion in Western blot analysis, as well as flow cytometric analysis.Polyclonal Abs against hBD2, hBD3, and mBD4 allowed specificdetection of the b-defensin domains of these fusion proteins. Absagainst mBD14 are not commercially available.Observations suggested that CCR2 may be another Gi protein-

coupled receptor for hBD3 (G. Jin, personal communication).

FIGURE 2. Selective binding of hBD2:Ig, hBD3:Ig, mBD4:Ig, and mB14:Ig to human CCR2. For binding analysis, HEK293 cells stably transfected with

human CCR2 (A) or human CXCR4 (B) were incubated with 1 mg/ml of b-defensin fusion protein (solid line) or 1 mg/ml human IgG1 (shaded graph).

Wild-type HEK293 cells were incubated with 1 mg/ml of the respective b-defensin fusion protein (dashed line). Binding was detected using a PE-

conjugated anti-human IgG Ab. Data are representative of two or three independent experiments.

6690 INTERACTION OF b-DEFENSINS WITH CCR2-EXPRESSING CELLS

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Therefore, we investigated whether hBD2, as well as hBD3 andtheir mouse orthologs mBD4 and mBD14, uses CCR2 in additionto CCR6. We highlight the effects of these b-defensins, fused to

the Fc region of human IgG1, with CCR2. As we reported earlier,the interaction of mBD4:Ig and mBD14:Ig with CCR6 is speciesspecific, but hBD2:Ig and hBD3:Ig were able to bind to humanand mouse CCR6. In contrast, the binding of the mBD fusionproteins, as well as their human orthologs, to CCR2 is not speciesspecific, and they interact with human or mouse receptors.Recombinant human CCR2-expressing HEK293 cells showed di-rectional migration induced by the b-defensin fusion proteins,indicating functional interaction of hBD2, hBD3, mBD4, andmBD14 with CCR2. Primary human peripheral blood monocytesexpressing CCR2, but not CCR6, also demonstrated consistentmigration induced by all of the b-defensin fusion proteins usedin this study.In an effort to ascertain whether the b-defensins and

MCP-1 share the same receptor, their mutual desensitizing effectswere investigated. Preincubation of monocytes with humanMCP-1 abolished the migration induced by hBD fusion proteins.Conversely, preincubation with hBD fusion proteins abolishedmigration induced by human MCP-1, indicating that these pro-teins share the same chemotaxis-inducing receptor. In contrast, the

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FIGURE 3. Comparison of the chemotactic ef-

fect of b-defensin fusion proteins and human

MCP-1 on human CCR2/HEK293 cells. Migration

of human CCR2/HEK293 cells, human CXCR4/

HEK293 cells, or wild-type HEK293 cells induced

by human MCP-1 (A) or human SDF-1a (B).

Chemotaxis of chemokine receptor-expressing or

wild-type HEK293 cells induced by hBD2:Ig (C),

hBD3:Ig (D), mBD4:Ig (E), or mBD14:Ig (F). G,

Migration induced by human IgG1. Data are repre-

sentative of three independent experiments. pp ,0.01, one-way ANOVA with the Dunnett posttest

versus media control.

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FIGURE 4. Expression of CCR2, but not CCR6, on human peripheral blood

monocytes. Monocytes, isolated from human blood, were labeled with anti-

hCCR2-PEAb(A, opengraph)or anti-CCR6-FITCAb(B, opengraph)and isotype

control (shaded graph). Data are representative of three independent experiments.

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chemotactic response to human SDF-1a was not desensitized bypreincubation with hBDs or human MCP-1 (data not shown). ThehBD fusion proteins induced chemotaxis of human monocytes

similarly to synthetic hBDs. Furthermore, pretreatment of mono-cytes with these synthetic peptides inhibited migration induced byhBD2:Ig and hBD3:Ig, demonstrating the capacity of synthetic

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FIGURE 5. b-defensin fusion protein-induced chemotaxis of human

peripheral blood monocytes. A, Dose-dependent migration of human mono-

cytes induced by human MCP-1. Cell migration of monocytes preincubated

for 30 min with 10 ng/ml hBD2:Ig or hBD3:Ig. B, Migration of monocytes

induced by unfused human IgG1. Chemotaxis of untreated monocytes or

monocytes preincubated for 30 min with 10 ng/ml human MCP-1 induced

by hBD2:Ig (C), hBD3:Ig (D), mBD4:Ig (E), or mBD14:Ig (F). Data are

representative of three or four independent experiments. pp , 0.01, one-

way ANOVAwith the Dunnett posttest versus media control.

medium

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FIGURE 6. Inhibition of b-defensin fusion protein-induced chemotaxis of

human peripheral blood monocytes by pretreatment with synthetic b-defen-

sins.Migration of humanmonocytes induced by 10 ng/ml hBD2:Ig, shBD2, or

unfused human IgG (A) and 10 ng/ml hBD3:Ig, shBD3, or unfused human IgG

(B).Abrogated chemotaxis ofmonocytes after pretreatment for 30minwith 10

ng/ml shBD2 in response to hBD2:Ig (A) and 10 ng/ml shBD3 in response to

hBD3:Ig (B). Data are representative of three independent experiments. pp,0.01, one-way ANOVAwith the Dunnett posttest versus media control.

100 101 102 103 104

FL1-H: anti-CCR6

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BA

FIGURE 7. Comparison of expression of CCR2 on PECs from wild-

type and CCR2-deficient C57BL/6 mice and expression of CCR6. A,

Thioglycollate-elicited mouse PECs from wild-type mice (solid line) or

CCR2-deficient mice (dashed line) labeled with anti-mCCR2 polyclonal

Ab from goat and secondary Ab anti-goat IgG-FITC and PECs incubated

with secondary Ab alone (shaded graph). B, PECs from wild-type mice

labeled with anti-mCCR6 (solid line) or isotype control (shaded graph).

Data are representative of three independent experiments.

0 0.1 1 10100

10000

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WT PECs

CCR2-/- PECs

A

mMCP-1 (ng/ml)

Mig

ratio

n (c

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PF)

mBD4:Ig 0 1 10100

100010000

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B

hIgG1 (ng/ml)

Mig

ratio

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r/H

PF)

0 1 10100

100010000

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*

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mBD4:Ig (ng/ml)

Mig

ratio

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PF)

0 1 10100

100010000

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35 WT PECs

CCR2-/- PECs

* *

* *

D

mBD14:Ig (ng/ml)

Mig

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n (c

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r/H

PF)

0 1 10100

100010000

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hBD2:Ig (ng/ml)

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hBD3:Ig (ng/ml)

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PF)

FIGURE 8. Comparison of migration of wild-type PECs and CCR2-

deficient PECs towardb-defensin fusion proteins.A, Chemotaxis of thioglycol-

late-elicited mouse PECs fromwild-type mice or CCR2-deficient mice induced

by human MCP-1. B, Migration of wild-type PECs induced by unfused human

IgG1. Chemotaxis induced by mBD4:Ig (C), mBD14:Ig (D), hBD2:Ig (E), or

hBD3:Ig (F). Data are representative of three or four independent experiments.

pp, 0.01, one-way ANOVAwith the Dunnett posttest versus media control.

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peptides to desensitize the interaction of recombinantly expressedfusion proteins with CCR2. The effects of synthetic mBD4 ormBD14 could not be tested, because these peptides are not com-mercially available. Additionally, chemoattraction of PECs fromC57BL/6 wild-type mice by human and mBD fusion proteins wasabrogated using cells from CCR2-deficient C57BL/6 mice. Be-cause the lack of CCR2 in these gene-targeted mice is specificand irreversible, we were able to evaluate CCR2-dependent che-motaxis without concern for residual receptor function and, thus,ruled out the involvement of another chemotaxis-inducing receptor.These results clearly demonstrate specific interaction of hBD2:Ig,hBD3:Ig, mBD4:Ig, and mBD14:Ig with human and mouse CCR2.A recent study indicated a possible interaction of hBD3 with the

chemokine receptor CXCR4. The investigators described thecompetition of hBD3 with SDF-1a binding to CXCR4 and furtherreported hBD3-induced internalization of the chemokine receptor.However, direct interaction with CXCR4 was not shown (22). Noneof the b-defensin fusion proteins used in the present study demon-strated direct binding to human CXCR4 or chemoattraction of hu-man CXCR4-expressing HEK293 cells. This argues against anydirect interaction of these b-defensin fusion proteins with CXCR4.Selective interaction of b-defensins with CCR2 and CCR6 raises

the question of the underlying common binding motif. Althoughb-defensins and CC-chemokines, such as CCL2/MCP-1 orCCL20/MIP-3a, do not share significant sequence similarities,they do share structural similarities, such as the a helix, theabundance of cationic residues, and disulfide linkages, responsiblefor the distinct tertiary structure. Thus, common structural motifsand electrostatic overlap may be responsible for the CC-chemokinereceptor-dependent chemotaxis-inducing activity of certainb-defensins (32). Although some chemokines also interact withmultiple receptors, and because no chemokines interact with bothCCR2 and CCR6, further studies are necessary to understand howb-defensins interact with several distinct chemokine receptors.CCR2 and CCR6 were shown to be crucial for the recruitment of

professional APCs to inflamed tissue (33). In particular, CCR2+

monocytes are recruited to sites of tissue inflammation, contrib-uting to clearing pathogens and triggering adaptive immune re-sponse, whereas CCR22 monocytes infiltrate noninflamed tissues,where they may be involved in tissue homeostasis (24). Ourfindings support a potential role for b-defensins as mediators ofenhanced immune reaction during inflammation. CCR6 isexpressed by different lymphocyte subsets, such as CD45RO+

CD4+ T cells (34) and Th17 cells (35), as well as immature den-dritic cells (14). Chemoattraction of these cells to sites of infectionis necessary to initiate the adaptive immune response. However,CCR2 is largely expressed on myeloid cells, such as monocytes,macrophages (36, 37), and neutrophils (38), which are crucial forinflammation and phagocytosis. Thus, our data suggest thatb-defensins contribute to the recruitment of a broad spectrum ofleukocytes to sites of infection and inflammation.In addition to their chemotactic and antimicrobial activity, there

is evidence suggesting that b-defensins participate in the regula-tion of host innate and adaptive immune responses. A recent studyprovided data showing the capacity of hBD1 to induce the expres-sion of maturation markers (e.g., MHC class II and CD83), cos-timulatory markers (e.g., CD80, CD86, and CD40), andproinflammatory cytokines (e.g., TNF, IL-6, and IL-12p70) inhuman monocyte-derived dendritic cells (39). Funderburg et al.(13) described TLR1- and TLR2-mediated expression of costimu-latory molecules on monocytes and myeloid dendritic cells in-duced by hBD3. Furthermore, mBD2 is able to induce TLR4-mediated maturation of dendritic cells (12). Thus, we hypothesizethat b-defensins, expressed by epithelial cells and leukocytes at

sites of infection and inflammation, promote chemotactic recruit-ment and activation of leukocytes, such as monocytes, macro-phages, and dendritic cells. Consequently, b-defensins producedat nanomolar concentrations by epithelial cells and leukocytes inresponse to infectious and injurious challenges form a haptotacticgradient in the tissue and along blood vessel walls to attract moreAPCs and monocytes to the endangered site. In addition, micro-molar concentrations of b-defensins exert their antimicrobial ac-tivity against invasive microorganisms.Recent studies indicated that CCR2+ macrophages are crucial

mediators of ulcerative colitis-associated colon carcinogenesis(40). In oral carcinoma, hBD3 was shown to be overexpressedby tumor cells, correlating with the recruitment and infiltrationof macrophages (41). Furthermore, endothelial progenitor cellswere reported to be recruited into tumors in a CCR2- andCCR5-dependent manner, contributing to neovascularization(42). These findings, together with our results, make it plausiblefor b-defensins to contribute to tumorigenesis and tumor develop-ment, as suggested by G. Jin (personal communication). Furtherstudies are required to identify potentially different cellular-recruitment patterns in tissues following infection, inflammation,or tumorigenesis, depending on the type of b-defensin expressed.CCR2 seems to be the unidentified Gi protein-coupled receptor

responsible for CCR6-independent b-defensin–mediated chemo-taxis of monocytes. The b-defensin fusion proteins are able torecruit a broad spectrum of leukocytes in a CCR6- and CCR2-dependent manner. Based on our data, we conclude that the re-cruitment capabilities of b-defensins contribute to the innate andadaptive immune response in their role as chemoattractants.

AcknowledgmentsWe thank Dr. O.M. Zack Howard for advice and discussions. We acknowl-

edge Anna Trivett and Steve Stull for technical assistance.

DisclosuresThe authors have no financial conflicts of interest.

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